Methods for detecting reverse triiodothyronine by mass spectrometry

ABSTRACT

Provided are methods for determining the amount of reverse T3 in a sample using mass spectrometry. The methods generally involve ionizing reverse T3 in a sample and detecting and quantifying the amount of the ion to determine the amount of reverse T3 in the sample.

FIELD OF THE INVENTION

The invention relates to the detection of reverse triiodothyronine. In aparticular aspect, the invention relates to methods for detectingreverse triiodothyronine by mass spetrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Reverse triiodothyronine((2S)-2-amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3-iodophenyl]propanoicacid) (rT3) is a non-active isomer of triiodothyronine (T3). T3 and rT3are both derived from thyoxine (T4) through the action of deiodinase asfollows:

Both T3 and rT3 bind to thyroid hormone receptors. When T3 binds, thereceptors are stimulated, thus increasing metabolic activity. Uponbinding, rT3, unlike T3, does not stimulate thyroid hormone receptors.Thus, rT3 does not stimulate metabolic activity of the target cell, andin fact, blocks receptor sites from T3 activation.

An excess if rT3 may result in widespread shutdown in T3 binding, acondition called reverse T3 dominance. Reverse T3 dominance results inreduced body temperature, which slows the action of many enzymes,leading to a clinical syndrome, Multiple Enzyme Dysfunction, whichproduces the effects seen in hypothyroidism.

Further, the process of 5′ monodeiodination that converts T4 to T3, andrT3 to diiodothyronine (DIT) is inhibited in a wide variety ofconditions, including fasting, malnutrition, poorly controlled diabetesmellitus, trauma, surgery, and systemic illness. Consequently, the serumT3 level typically decreases, and the rT3 level often increases in thesecircumstances. Thus, the ratio of T3 to rT3 is an important diagnosticmarker for the metabolism and function of thyroid hormones and relatedcompounds in clinical chemistry.

Assays for T4, T3, and related compounds (including rT3) have beendeveloped and are used to evaluate thyroid status or to optimizetherapeutic dosages. Assay formats include radioimmunoassay and massspectrometry. For example, Hantson et al. reported quantitatingderivatized thyroid hormones via GC-MS (Hansen et al., J. Chromatogr. B(2004), 807:185-192); Zhang et al. reported quantitating T3 and rT3 inhuman serum via SPE-ESI-MS/MS (Zhang et al., J. Am. Soc. Mass Spectrom.(2005), 16:1781-86); Tai et al. reported quantitating T3 in serum viaSPE-HPLC-MS/MS (Tai et al., Anal. Chem. (2004), 76:5092:96); Couldwellet al. report mass spectrometric analysis, including fragmentationspectra, of rT3 in standard organic solvents by ESI-MS/MS (Couldwell etal., Rapid Comm. Mass Spectrom. (2005), 19:2295-2304); Wang andStapleton report quantitation of rT3 in spiked bovine serum samples viaSPE-LC-ESI-MS/MS (Wang and Stapleton, Anal Bioanal Chem (2010),397:1831-39).

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount ofreverse T3 (rT3) in a sample by mass spectrometry, including tandem massspectrometry.

In one aspect, methods are provided for determining the amount of rT3 ina body fluid sample by mass spectrometry. Methods of this aspectinclude: (a) ionizing rT3 from the body fluid sample to produce one ormore rT3 ions detectable by mass spectrometry; and (b) detecting theamount of the rT3 ion(s) by mass spectrometry. Once the amount of theone or more rT3 ions is measured, the amount of rT3 ion(s) determined isrelated to the amount of rT3 in the body fluid sample. In some methodsof the present invention, rT3 from the body fluid sample is notsubjected to solid phase extraction prior to ionization.

In some embodiments, rT3 from the body fluid sample is subjected toliquid chromatography prior to being ionized. In some embodiments, theliquid chromatography comprises high performance liquid chromatography(HPLC).

In some embodiments, rT3 from the body fluid sample is enriched byprotein precipitation prior to being ionized. In some embodiments, theprotein precipitation is conducted prior to liquid chromatography. Insome embodiments, protein precipitation is conducted by contacting thebody fluid sample with an organic solvent in an amount sufficient toprecipitate at least a portion of protein that may be present in thebody fluid sample. In some related embodiments, the organic solventcomprises methanol.

In some embodiments, methods determining the amount of reverse T3 (rT3)in a body fluid sample by mass spectrometry are provided which includeprocessing a body fluid sample to generate a processed sample comprisingrT3 from a body fluid sample. In related methods, the processingcomprises: i) precipitating protein from the body fluid sample by addingan organic solvent, such that the resulting supernatant comprises theorganic solvent and rT3 from the body fluid sample; ii) purifying rT3 inthe supernatant by subjecting the supernatant to a reverse-phase highperformance liquid chromatography (RP-HPLC) column, wherein thepurifying comprises introducing an aqueous solution to the columnimmediately prior to introducing the supernatant; and iii) eluting rT3from the RP-HPLC column to generate a processed sample comprising rT3.This processed sample may then be analyzed as described above; namely,by ionizing rT3 in the processed sample to generate one or more reverseT3 ions detectable by mass spectrometry; determining the amount of oneor more rT3 ions by mass spectrometry; and using the amount of thedetermined rT3 ions to determine the amount of rT3 in the body fluidsample. In some embodiments, the organic solvent comprises methanol. Insome related embodiments, the supernatant generated in step ii)comprises at least 10% methanol

In embodiments where an aqueous plug is introduced into an RP-HPLCcolumn prior to introduction of an rT3-containing sample, the ratio ofthe sample volume to the aqueous plug volume may be within the range ofabout 10:1 to about 1:10; such as within the range of about 5:1 to about1:5; such as about 1:1.

In some embodiments, one or more rT3 ions detectable by massspectrometry are selected from the group consisting of ions with amass/charge ratio of 649.9±0.5, 605.2±0.5, and 127.1±0.5. In someembodiments, the ions are selected from the group consisting of ionswith a mass/charge ratio of 649.9±0.5 and 605.2±0.5.

In some embodiments, mass spectrometry comprises tandem massspectrometry. In some related embodiments, one or more rT3 ionsdetectable by mass spectrometry comprise a precursor ion with amass/charge ratio of 649.9±0.5, and a fragment ion selected from thegroup of ions with a mass/charge ratio of 605.2±0.5 and 127.1±0.5. Insome embodiments, the fragment ion has a mass/charge ratio of 605.2±0.5.

In some embodiments, the body fluid sample comprises plasma or serum,such as plasma or serum taken from a human. In some related embodiments,the methods described herein may be used to determine the amount of rT3present in a plasma or serum sample when taken from a human.

In certain embodiments of the methods disclosed herein, massspectrometry is performed in negative ion mode. Alternatively, massspectrometry is performed in positive ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI) or electrospray ionization (ESI), may be used in embodiments ofthe present invention. In certain embodiments, rT3 is measured using ESIin negative ion mode.

In some embodiments, a separately detectable internal rT3 standard isprovided in the sample, the amount of which is also determined in thesample. In these embodiments, all or a portion of both the endogenousrT3 and the internal standard present in the sample is ionized toproduce a plurality of ions detectable in a mass spectrometer, and oneor more ions produced from each are detected by mass spectrometry.

A preferred internal rT3 standard is ¹³C₆-rT3. In preferred embodiments,the internal rT3 standard ions detectable in a mass spectrometer areselected from the group consisting of negative ions with m/z of655.8±0.50, 611.1±0.50, and 127.1±0.50. In embodiments utilizing tandemmass spectrometry, ¹³C₆-rT3 ions may comprise a precursor ion with m/zof 655.8±0.50 and a fragment ion with m/z of 611.1±0.50.

In preferred embodiments, the presence or amount of the rT3 ion isrelated to the presence or amount of rT3 in the test sample bycomparison to a reference such as ¹³C₆-rT3.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedrT3 parent or daughter ions by mass spectrometry. Relative reduction asthis term is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “test sample” refers to any sample that maycontain rT3. As used herein, the term “body fluid” means any fluid thatcan be isolated from the body of an individual. For example, “bodyfluid” may include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), ultra high performance liquidchromatography (UPLC), and high turbulence liquid chromatography (HTLC).

As used herein, the term “high performance liquid chromatography” or“HPLC” refers to liquid chromatography in which the degree of separationis increased by forcing the mobile phase under pressure through astationary phase on a support matrix, typically a densely packed column.As used herein, the term “ultra high performance liquid chromatography”or “UPLC” or “UHPLC” (sometimes known as “ultra high pressure liquidchromatography”) refers to HPLC which is conducted at higher pressuresthan traditional HPLC techniques (ca.>5000 psi) and optionally withcolumn packing materials with smaller particle sizes (ca.<5 μm).

As used herein, the term “high turbulence liquid chromatography” or“HTLC” refers to a form of chromatography that utilizes turbulent flowof the material being assayed through the column packing as the basisfor performing the separation. HTLC has been applied in the preparationof samples containing two unnamed drugs prior to analysis by massspectrometry. See, e.g., Zimmer et al., J. Chromatogr. A 854: 23-35(1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and5,772,874, which further explain HTLC. Persons of ordinary skill in theart understand “turbulent flow”. When fluid flows slowly and smoothly,the flow is called “laminar flow”. For example, fluid moving through anHPLC column at low flow rates is laminar. In laminar flow the motion ofthe particles of fluid is orderly with particles moving generally instraight lines. At faster velocities, the inertia of the water overcomesfluid frictional forces and turbulent flow results. Fluid not in contactwith the irregular boundary “outruns” that which is slowed by frictionor deflected by an uneven surface. When a fluid is flowing turbulently,it flows in eddies and whirls (or vortices), with more “drag” than whenthe flow is laminar. Many references are available for assisting indetermining when fluid flow is laminar or turbulent (e.g., TurbulentFlow Analysis: Measurement and Prediction, P. S. Bernard & J. M.Wallace, John Wiley & Sons, Inc., (2000); An Introduction to TurbulentFlow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 35 μm. As used in this context, the term“about” means±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%. In apreferred embodiment the analytical column contains particles within therange of about 1.5 to about 5 μm in diameter, such as about 2.6 μm indiameter.

As used herein, the term “on-line” or “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. Nos. 6,204,500,entitled “Mass Spectrometry From Surfaces;” 6,107,623, entitled “Methodsand Apparatus for Tandem Mass Spectrometry;” 6,268,144, entitled “DNADiagnostics Based On Mass Spectrometry;” 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis21:1164-67 (2000).

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber. As the droplets get smaller the electrical surface chargedensity increases until such time that the natural repulsion betweenlike charges causes ions as well as neutral molecules to be released.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. Robb, D. B., Covey, T. R. and Bruins,A. P. (2000): See, e.g., Robb et al., Atmospheric pressurephotoionization: An ionization method for liquid chromatography-massspectrometry. Anal. Chem. 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LLOQ isidentifiable, discrete and reproducible with a concentration at whichthe standard deviation (SD) is less than one third of the totalallowable error (TEa; arbitrarily set for rT3 as 30% of the LLOQ).

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as the mean of the blankplus four times the standard deviation of the blank.

As used herein, an “amount” of rT3 in a body fluid sample refersgenerally to an absolute value reflecting the mass of rT3 detectable involume of body fluid. However, an amount also contemplates a relativeamount in comparison to another rT3 amount. For example, an amount ofrT3 in a body fluid can be an amount which is greater than a control ornormal level of rT3 normally present.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B show schematic diagrams of HPLC pump configurations whichresult in introduction of an aqueous plug immediately prior tointroduction of the sample. Aqueous solvents are shown in black, whilethe sample with high organic solvent content is shown in grey. FIG. 1Ashows the loading phase (i.e., loading of a sample loop). FIG. 1B showsordered introduction of the fluid plugs into the HPLC.

FIGS. 2A and B show exemplary chromatograms for. T3 and rT3 inmethanol-based samples collected by HPLC-MS/MS. The chromatograms werecollected with (FIG. 2A) and without (FIG. 2B) introduction of anaqueous plug to the HPLC immediately prior to introduction of 100 μL ofsample. Details are discussed in Example 3.

FIG. 3 shows exemplary chromatograms for T3 and rT3 in an acetone-basedsample collected by HPLC-MS/MS. The chromatograms were collected withoutintroduction of an aqueous plug to the HPLC immediately prior tointroduction of 100 μL of sample. Details are discussed in Example 3.

FIGS. 4A and B show exemplary chromatograms of rT3 and ¹³C₆-rT3(internal standard), respectively. Details are discussed in Example 5.

FIG. 5 shows a typical calibration curve generated by analyzingcalibration samples with rT3 from 25 pg/mL to 2000 pg/mL. Details aredescribed in Example 6.

FIG. 6 shows a plot of data generated in lower limit of quantitation(LLOQ), limit of detection (LOD), and limit of blank (LOB) experiments.Details are described in Example 9.

FIG. 7 shows linearity of rT3 detection to at least about 200 ng/dL.Details are described in Example 10.

FIGS. 8A and B show comparison and difference plots, respectively, ofrT3 quantitation in EDTA plasma and serum. Details are described inExample 11.

FIGS. 9A and B show comparison and difference plots, respectively, ofrT3 quantitation in Heparin plasma and serum. Details are described inExample 11.

FIGS. 10A and B show comparison and difference plots, respectively, ofrT3 quantitation in SST serum and serum. Details are described inExample 11.

DETAILED DESCRIPTION OF THE INVENTION

Methods of the present invention are described for measuring the amountof rT3 in a sample. More specifically, mass spectrometric methods aredescribed for detecting and quantifying rT3 in a sample. The methods mayutilize liquid chromatography (LC), most preferably HPLC or UPLC, toperform a purification of selected analytes, and combine thispurification with unique methods of mass spectrometry (MS), therebyproviding a high-throughput assay system for detecting and quantifyingrT3 in a test sample. The preferred embodiments are particularly wellsuited for application in large clinical laboratories for automated rT3assay. The methods provided are accomplished without the necessity ofsample purification via solid phase extraction prior to liquidchromatography.

Suitable samples for use in methods of the present invention include anysample that may contain the analyte of interest. In some preferredembodiments, a sample is a biological sample; that is, a sample obtainedfrom any biological source, such as an animal, a cell culture, an organculture, etc. In certain preferred embodiments, samples are obtainedfrom a mammalian animal, such as a dog, cat, horse, etc. Particularlypreferred mammalian animals are primates, most preferably male or femalehumans. Particularly preferred samples include bodily fluids such asblood, plasma, serum, saliva, cerebrospinal fluid, or a tissue sample.Such samples may be obtained, for example, from a patient; that is, aliving person, male or female, presenting oneself in a clinical settingfor diagnosis, prognosis, or treatment of a disease or condition. Thesample is preferably obtained from a patient, for example, blood serumor plasma.

The present invention contemplates kits for a rT3 quantitation assay. Akit for a rT3 quantitation assay of the present invention may include akit comprising an internal standard, in an amount sufficient for atleast one assay. Typically, the kits will also include instructionsrecorded in a tangible form (e.g., contained on paper or an electronicmedium) for using the packaged reagents for use in a measurement assayfor determining the amount of rT3.

Calibration and QC pools for use in embodiments of the present inventioncan be prepared using “stripped” plasma or serum (stripped of rT3): forexample, analyte-stripped, defibrinated and delipidized plasma/serum.All sources of human or non-human plasma or stripped serum should bechecked to ensure that they do not contain measurable amounts ofendogenous rT3.

Sample Preparation for Mass Spectrometry

Various methods may be used to enrich rT3 relative to other components(e.g. protein) in the sample prior mass spectrometry, including forexample, liquid chromatography, filtration, centrifugation, thin layerchromatography (TLC), electrophoresis including capillaryelectrophoresis, affinity separations including immunoaffinityseparations, extraction methods including ethyl acetate extraction andmethanol extraction, and the use of chaotropic agents or any combinationof the above or the like.

Protein precipitation is one preferred method of preparing a sample,especially a biological sample, such as serum or plasma. Proteinprecipitation may be used to remove at least a portion of the proteinpresent in a sample leaving rT3 in the supernatant. Precipitated samplesmay be centrifuged to separate the liquid supernatant from theprecipitated proteins; alternatively the samples may be filtered, forexample through a glass fiber filter, to remove precipitated proteins.The resultant supernatant or filtrate may then be applied directly tomass spectrometry analysis; or alternatively to liquid chromatographyand subsequent mass spectrometry analysis.

Various precipitation agents are known in the art, such as acetone,alcohols such as methanol, or various acidifying agents. In certainembodiments, the use of protein precipitation such as for example,methanol protein precipitation, may obviate the need for solid phaseextraction (SPE) such as high turbulence liquid chromatography (HTLC),or other on-line extraction prior to mass spectrometry, or HPLC or UPLCand mass spectrometry.

Accordingly, in some embodiments, the method involves (1) performing aprotein precipitation of the sample of interest; and (2) loading thesupernatant directly onto the LC-mass spectrometer without using SPE.

In other embodiments, HTLC, alone or in combination with one or morepurification methods, may be used to purify rT3 prior to massspectrometry. In such embodiments samples may be extracted using an HTLCextraction cartridge which captures the analyte, then eluted andchromatographed on a second HTLC column or onto an analytical HPLC orUPLC column prior to ionization. Because the steps involved in thesechromatography procedures may be linked in an automated fashion, therequirement for operator involvement during the purification of theanalyte can be minimized. This feature may result in savings of time andcosts, and eliminate the opportunity for operator error.

According to some embodiments, the method involves protein precipitationfrom serum or plasma samples. In these embodiments, a reagent whichcauses proteins to precipitate out of serum or plasma, such as methanol,acetonitrile, isopropanol, acetone, or zinc sulfate solution may beadded, along with internal standard, to the sample in quantitiessufficient to precipitate proteins from the sample. For example,methanol may be added to serum samples at a ratio within the range ofabout 1:1 to about 10:1; such as about 2:1 to about 5:1; such as about3:1. After the proteins have been precipitated, the mixtures may then becentrifuged, with rT3 remaining in the supernatant. The supernatant maythen be collected and subjected to mass spectrometric analysis, with orwithout further purification.

One additional such means of sample purification that may be used priorto mass spectrometry is liquid chromatography (LC). Liquidchromatography, including high-performance liquid chromatography (HPLC),relies on relatively slow, laminar flow technology. Traditional HPLCanalysis relies on column packing in which laminar flow of the samplethrough the column is the basis for separation of the analyte ofinterest from the sample. The skilled artisan will understand thatseparation in such columns is a diffusional process and may select HPLCinstruments and columns that are suitable for use with rT3. Thechromatographic column typically includes a medium (i.e., a packingmaterial) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlesinclude a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the chemical moieties. One suitablebonded surface is a hydrophobic bonded surface such as an alkyl bondedsurface. Alkyl bonded surfaces may include C-4, C-8, C-12, or C-18bonded alkyl groups, preferably C-18 bonded groups. The chromatographiccolumn includes an inlet port for receiving a sample directly orindirectly (such as from a coupled SPE column) and an outlet port fordischarging an effluent that includes the fractionated sample.

In one embodiment, the sample may be applied to the column at the inletport, eluted with a solvent or solvent mixture, and discharged at theoutlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In some embodiments, HPLC or UPLC is conducted with a hydrophobic columnchromatographic system. In certain embodiments, a C18 analytical column(e.g., a Kinetex C18 with TMS endcapping analytical column fromPhenomenex (2.6 μm particle size, 50×4.6 mm), or equivalent) is used. Incertain embodiments, HTLC and/or HPLC and/or UPLC are performed usingHPLC Grade 0.1% aqueous formic acid and 100% methanol as the mobilephases.

Reverse phase HPLC is generally conducted with a non-polar stationaryphase and an aqueous, moderately polar mobile phase. Under theseconditions, samples injected for analysis which contain a large organicor alcohol solvent content pass over the stationary phase of the columnwithout significant interaction, leading to poor column performance(i.e., less analyte retention and poor peak shape). One of twostrategies is typically employed to counteract this effect. First, thesamples comprising a high organic or alcohol content (such as thosegenerated by alcohol protein precipitation) may be dried andreconstituted in a predominantly aqueous solvent. Second, very smallvolumes of samples comprising a high organic or alcohol content may beused, with the expectation that effects of such small absolute organicor alcohol volumes will largely be overcome because of the relativevolumes of mobile phase to sample volume. Both approaches havesignificant detractors for clinical laboratory assays. Drying andreconstituting samples adds significant time and expense to what mayotherwise be automated procedures, while use of very small samplevolumes may diminish assay sensitivity by limiting the amount of analyteintroduced to the column.

The present invention provides methods to overcome the above describedcomplications. It has been found that a “plug” of aqueous or mostlyaqueous solvent introduced to a reverse phase HPLC column immediatelyprior to introduction of a sample with a high organic or alcohol contentavoids problems associated with such samples. The present methods may beapplied to samples with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 100% (v/v) organic or alcohol, or mixtures thereof. In someembodiments, the sample solvent is methanol. For typical commerciallyavailable reverse-phase HPLC columns, an aqueous plug volume of about 10μL to 1000 μL may be introduced immediately prior to about 10 μL to 1000μL of a sample. Preferably the ratio of plug volume to sample volumewill be in the range of about 5:1 to about 1:5; such as within the rangeof about 2:1 to about 1:2; such as about 1:1. Appropriate absolute andrelative volumes of each solution will vary with variables such as theorganic solvent content of the sample, the concentration of the analytein the sample, column packing material, and column volume. However, itis within the skill of one skilled in the art to determine appropriateabsolute and relative volumes of each solution.

The artisan will recognize that there are numerous ways to achieve theordered introduction of multiple solutions onto an HPLC column usingvarious configurations of plumbing and pumps. In some embodiments, asample loop of a predetermined volume is used to achieve the orderedintroduction of an aqueous plug, such as a plug that has no organicsolvent component (i.e., a plug with a purely aqueous solventcomponent), prior to introduction of a sample comprising a high organicor alcohol solvent content. In these embodiments, the sample loop isinitially filled with an aqueous fluid to capacity. A volume of organicor alcohol containing sample is then introduced into the sample loopsuch that the loop is only partially occupied by the organic or alcoholcontaining sample while at least some aqueous fluid remains in the loop.Then, a series of valves and pumps, or other plumbing components, isused to direct the aqueous plug followed by the organic or alcoholcontaining sample from the sample loop onto the HPLC column. FIGS. 1Aand 1B show schematic representations of such a system in operation.

Once the analyte has been eluted from a first chromatography column, itmay be subjected to further chromatography on one or more additionalcolumns. By careful selection of valves and connector plumbing, two ormore chromatography columns may be connected as needed such thatmaterial is passed from one to the next without the need for any manualsteps. In preferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

Detection and Quantitation by Mass Spectrometry

In various embodiments, rT3 present in a sample may be ionized by anymethod known to the skilled artisan. Mass spectrometry is performedusing a mass spectrometer, which includes an ion source for ionizing thefractionated sample and creating charged molecules for further analysis.For example ionization of the sample may be performed by electronionization, chemical ionization, electrospray ionization (ESI), photonionization, atmospheric pressure chemical ionization (APCI),photoionization, atmospheric pressure photoionization (APPI), fast atombombardment (FAB), liquid secondary ionization (LSI), matrix assistedlaser desorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), inductively coupled plasma (ICP) and particle beamionization. The skilled artisan will understand that the choice ofionization method may be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

In preferred embodiments, rT3 is ionized by heated electrosprayionization (ESI) in negative mode.

In mass spectrometry techniques generally, after the sample has beenionized the positively charged or negatively charged ions therebycreated may be analyzed to determine a mass-to-charge ratio. Suitableanalyzers for determining mass-to-charge ratios include quadrupoleanalyzers, ion trap analyzers, magnetic and electric sector analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected, i.e. usinga selective ion monitoring mode (SIM), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM). Preferably, the mass-to-chargeratio is determined using a quadrupole analyzer. For example, in a“quadrupole” or “quadrupole ion trap” instrument, ions in an oscillatingradio frequency field experience a force proportional to the DCpotential applied between electrodes, the amplitude of the RF signal,and the mass/charge ratio. The voltage and amplitude may be selected sothat only ions having a particular mass/charge ratio travel the lengthof the quadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, standards may be run with the samples,and a standard curve constructed based on ions generated from thosestandards. Using such a standard curve, the relative abundance of agiven ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of rT3.Methods of generating and using such standard curves are well known inthe art and one of ordinary skill is capable of selecting an appropriateinternal standard. For example, an isotopically labeled rT3 may be usedas an internal standard; in certain preferred embodiments the standardis ¹³C₆-rT3. Numerous other methods for relating the amount of an ion tothe amount of the original molecule will be well known to those ofordinary skill in the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation is oftenused to generate the fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, rT3 is detected and/or quantifiedusing MS/MS as follows. Samples are subjected to protein precipitationfollowed by liquid chromatography, preferably HPLC or UPLC; the flow ofliquid solvent from the liquid chromatography column enters an ESInebulizer interface of an MS/MS analyzer; and the solvent/analytemixture is converted to vapor in the heated tubing of the interface. Theanalyte (e.g., rT3), contained in the nebulized solvent, is ionized assolvent present in the nebulized droplets is vaporized. The ions, e.g.precursor ions, pass through the orifice of the instrument and enter thefirst quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters,allowing selection of ions (i.e., selection of “precursor” and“fragment” ions in Q1 and Q3, respectively) based on their mass tocharge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ionsare fragmented. The first quadrupole of the mass spectrometer (Q1)selects for molecules with the mass to charge ratios of rT3. Precursorions with the correct mass/charge ratios are allowed to pass into thecollision chamber (Q2), while unwanted ions with any other mass/chargeratio collide with the sides of the quadrupole and are eliminated.

Precursor ions entering Q2 collide with neutral collision gas moleculesand fragment. This process is called collision activated dissociation(CAD). The fragment ions generated are passed into quadrupole 3 (Q3),where the fragment ions of rT3 are selected while other ions areeliminated. In some embodiments, rT3 precursor ions are fragmented viacollision with an inert collision gas such as argon or nitrogen,preferably nitrogen.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably negative ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of rT3 that may be usedfor selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte ofinterest. In certain embodiments, the area under the curves, oramplitude of the peaks, for fragment ion(s) and/or precursor ions aremeasured to determine the amount of rT3. As described above, therelative abundance of a given ion may be converted into an absoluteamount of the original analyte, e.g., rT3, using calibration standardcurves based on peaks of one or more ions of an internal molecularstandard, such as ¹³C₆-rT3.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1 Sample (Serum) and Reagent Preparation

Serum samples were prepared by collecting blood in a standard red-topserum Vacutainer® tube and allowed to clot at room temperature for 30minutes. Samples were then centrifuged and the serum separated from thecells immediately. Alternately, blood was collected in a double-gelbarrier tube, allowed to clot at room temperature. Samples were thencentrifuged and the serum separated from the cells within 24 hours.

Plasma samples collected in EDTA plasma Vacutainer® tubes and sodiumheparin Vacutainer® tubes were also prepared for analysis.

Three rT3 stock solutions were prepared. An initial rT3 stock solutionof 1 mg/mL in methanol/basic solution was prepared by dissolving rT3 in40 mL concentrated NaOH diluted to 100 mL with methanol. An intermediatestock solution of 1,000,000 pg/mL rT3 was prepared by further diluting aportion of the initial stock solution with methanol. Finally, a workingstock solution of 10,000 pg/mL rT3 was prepared by further diluting aportion of the intermediate stock solution with double-stropped charcoalserum.

¹³C₆-rT3 internal standard solutions were prepared similarly to the rT3solutions described above, except that the final working ¹³C₆-rT3internal standard was prepared to a final concentration of 500 pg/mL bydilution with methanol rather than stripped serum.

Example 2 Enrichment of rT3 in Serum by Protein Precipitation

100 μL of specimens were first added to a well in a 96 well plate. 300μL of the 500 pg/mL ¹³C₆-rT3 in methanol solution (internal standard)was then added to each well, with each well checked for precipitateformation. After visually confirming precipitation, the well plate wasmixed for about 1 minute at about 1500 rpm, allowed to rest, mixedagain, refrigerated for about 30 minutes, and mixed a final time. Afterthe final mix, the plate was centrifuged at a minimum of 3000×g for atleast 30 minutes.

Example 3 Comparison of HPLC-MS/MS of rT3 in Methanol Solution with andwithout Leading Aqueous Plug

Samples containing rT3 were prepared as indicated in Example 2 viamethanol precipitation and via a similar procedure with acetoneprecipitation. The resulting samples contained a relatively high percentmethanol or acetone as solvent.

100 μL of the methanol-solvent based samples were analyzed with andwithout introduction of an aqueous plug of about 100 μL to an HPLCanalytical column (Phenomenex Kinetex C18 with TMS endcapping, 100×4.6mm, 2.6 μm particle size column) immediately prior to introduction ofthe sample. Mass chromatograms collected for both conditions are seen inFIGS. 2A-B. For comparison, 100 μL of an acetone-solvent based samplewas also analyzed without introduction of an aqueous plug. An exemplarymass chromatogram for the acetone-based sample is seen in FIG. 3.

As seen in FIGS. 2A-B and 3, the ion signal intensity for both T3 andrT3 was greatly enhanced for the sample purified via HPLC followingintroduction of an aqueous plug.

Example 4 Enrichment of rT3 Liquid Chromatography

The supernatants resulting from the centrifugation in Example 2 weresubjected to high performance liquid chromatography for furtherenrichment of rT3 prior to mass spectrometric analysis. Sample injectionwas performed with a Cohesive Technologies Aria TLX-1 HTLC systemoperating in laminar flow mode using Aria OS V 1.5 or newer software.

The HTLC system automatically injected of 100 μL of the above preparedsupernatants into the analytical column (Phenomenex Kinetex C18 with TMSendcapping, 100×4.6 min, 2.6 μm particle size column). A binary HPLCgradient was applied to the analytical column, to separate rT3 fromother analytes contained in the sample. Mobile phase A was 0.1% aqueousformic acid and mobile phase B was 100% methanol. The HPLC gradientstarted with a mixture of 70% mobile phase A and 30% mobile phase B, andwas ramped to 5% mobile phase A and 95% mobile phase B over 300 seconds.This ratio was then held for an additional 60 seconds, before beingreturned to the original mixture for 60 seconds. Under these conditions,rT3 (and ¹³C₆-rT3) eluted off of the HPLC column at approximately 235seconds. The eluted analytes were then subjected to MS/MS forquantitation.

Example 5 Detection and Quantitation of rT3 by MS/MS

MS/MS was performed using an ABSciex 5500 MS/MS system (ABSciex). Thefollowing software programs all from ABSciex were used in the Examplesdescribed herein: Analyst 1.4 or newer. Liquid solvent/analyte exitingthe analytical HPLC column flowed to the ESI interface of the MS/MSanalyzer. The solvent/analyte mixture was converted to vapor upon exitfrom the tubing of the interface. Analytes in the nebulized solvent wereionized by ESI in negative ion mode. Exemplary mass spectrometerparameters are shown in Table 1.

TABLE 1 Mass Spectrometer Operating Parameters Parameter Value ParameterValue Curtain Gas 30.0 Declustering −100.0 V  Potential Collision Gas 8Entrance −10.0 V Potential IonSpray Voltage −2500 V Collision Energy−40.0 V Temperature 700.0° C. Exit Lens    10 V Ion Source Gas 1 70.0Collision Cell −10.0 V Exit Potential Ion Source Gas 2 40.0

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of 649.9±0.50 for rT3 and 655.8±0.50 for ¹³C₆-rT3.Ions entering Quadrupole 2 (Q2) collided with nitrogen gas to generateion fragments, which were passed to quadrupole 3 (Q3) for furtherselection. Simultaneously, the same process using isotope dilution massspectrometry was carried out with an internal standard, ¹³C₆-rT3. Themass transitions used for detection and quantitation during validationon negative polarity are shown in Table 2. Additional mass transitionsof 649.9±0.50→127.1±0.50 and 655.8±0.50→127.1±0.50 were observed for rT3and ¹³C₆-rT3, respectively.

TABLE 2 Mass Transitions for rT3 (Negative Polarity) Analyte PrecursorIon (m/z) Product Ion (m/z) rT3 649.9 ± 0.50 605.2 ± 0.50 ¹³C₆-rT3(internal standard) 655.8 ± 0.50 611.1 ± 0.50

Exemplary chromatograms for rT3 and ¹³C₆-rT3 (internal standard)generated by monitoring the transitions shown in Table 2 are found inFIGS. 4A and B, respectively.

Example 6 Exemplary Calibration Curve Determination for rT3 by MS

Seven calibrator standards of rT3 in stripped serum at concentrations of25 pg/mL, 50 pg/mL, 100 pg/mL, 250 pg/mL, 500 pg/mL, 1000 pg/mL, and2000 pg/mL were prepared and analyzed as outlined above to generate anexemplary calibration curve. One such calibration curve is demonstratedin FIG. 5. The calibration curve shown was analyzed by linearregression, resulting in the following coefficients: y=0.0117x+−0.00213,and r=0.9988.

Example 7 Tests for Interfering Substances

Samples containing triglycerides (up to about 2000 mg/dL), bilirubin (upto about 50 mg/dL), and/or hemoglobin (up to about 500 mg/dL) weretested for possible interferences. No interference from these substanceswas detected.

Example 8 rT3 Assay Precision and Accuracy

Three quality control (QC) pools were prepared by spiking rT3 instripped serum at 10 ng/dL, 25 ng/dL, and 100 ng/dL.

Five aliquots from each of the three QC pools were analyzed in each offive assays to determine the accuracy and coefficient of variation (CV(%)) of a sample within an assay. The data and results of theseexperiments are found in Table 3.

TABLE 3 rT3 Assay Precision and Accuracy Run 1 Run 2 Run 3 Run 4 Run 5Level 1 (10 ng/dL) 1 10.60 10.30 10.20 9.73 10.10 2 10.90 9.81 10.4010.50 10.00 3 10.20 9.63 9.39 9.77 9.67 4 10.00 10.00 10.00 9.67 9.45 510.60 9.58 10.60 10.50 9.97 Count 5 5 5 5 5 Average 10.46 9.86 10.1210.03 9.84 Within-Run (WR) SD 0.36 0.29 0.46 0.43 0.27 Level 2 (25ng/dL) 1 26.00 23.80 25.80 24.90 24.70 2 24.50 24.60 24.70 24.60 26.10 324.60 25.30 25.40 24.60 25.30 4 25.10 25.00 25.40 26.60 24.40 5 25.5024.00 24.80 25.00 23.90 Count 5 5 5 5 5 Average 25.14 24.54 25.22 25.1424.88 Within-Run (WR) SD 0.63 0.64 0.46 0.84 0.85 Level 3 (100 ng/dL) 1100.00 95.10 99.80 95.30 98.50 2 97.30 97.00 98.30 98.60 96.80 3 97.7095.30 101.00 103.00 96.50 4 102.00 95.10 102.00 97.90 97.40 5 94.2097.20 98.70 98.60 98.10 Count 5 5 5 5 5 Average 98.24 95.94 99.96 98.6897.46 Within-Run (WR) SD 2.95 1.06 1.55 2.77 0.84 Summary Level 1 Level2 Level 3 Count 25 25 25 Mean 10.06 24.98 98.06 Pooled WR SD 0.37 0.702.03 Pooled WR CV 3.68% 2.79% 2.07% Overall STD 0.41 0.69 2.30 OverallCV (%) 4.06% 2.75% 2.34% Target value 10 25 100 Accuracy (%) 100.6%99.9% 98.1%

As shown in Table 3, the accuracy and coefficient of variation (CV (%))at each QC level were acceptable for use as a clinical assay.

Example 9 Analytical Sensitivity: Limit of Blank (LOB), Limit ofDetection (LOD) and Lower Limit of Quantitation (LLOQ)

The LLOQ refers to the concentration where measurements becomequantitatively meaningful. The analyte response at the LLOQ isidentifiable, discrete and reproducible at a concentration at which thestandard deviation (SD) is less than one third of the total allowableerror (TEa; arbitrarily set for rT3 as 30% of the LLOQ). The LOD is theconcentration at which the measured value is larger than the uncertaintyassociated with it. The LOD is the point at which a value is beyond theuncertainty associated with its measurement and is defined as the meanof the blank plus four times the standard deviation of the blank. TheLOB is set as two standard deviations above the mean measured value fora zero calibration standard.

The LLOQ, LOD, and LOB were determined by assaying samples atconcentrations close to the expected LLOQ and determining thereproducibility (five replicates each at 0, 2, 4, and 8 ng/dL rT3assayed in five runs) then determining the standard deviation (SD). Theresults were plotted for rT3 (shown in FIG. 6). The LOB, LOD, and LLOQwere determined to be from the curves to be 0.309 ng/dL, 0.392 ng/dL,and 2.050 ng/dL, respectively. Data from these experiments are presentedin Table 4.

TABLE 4 rT3 Limit of Blank (LOB), Limit of Detection (LOD) and LowerLimit of Quantitation (LLOQ) Studies Pool A Pool B Pool C Zero Cal StdRun Result (2 ng/dL) (4 ng/dL) (8 ng/dL) (0 ng/dL) 1 1 2.330 3.870 7.9400.224 2 2.120 4.120 7.770 0.162 3 1.960 3.810 7.600 0.229 4 2.060 4.0908.470 0.171 5 1.990 4.190 8.070 0.282 2 1 2.010 4.230 8.100 0.252 22.090 3.910 8.530 0.216 3 2.170 4.340 8.440 0.251 4 1.780 3.850 7.5500.180 5 2.120 4.190 7.990 0.149 3 1 2.170 4.380 7.520 0.213 2 1.7803.670 7.460 0.187 3 2.080 3.920 8.310 0.222 4 2.100 4.170 7.720 0.191 52.080 4.720 7.680 0.255 4 1 2.240 4.410 7.800 0.245 2 1.710 3.880 8.6000.272 3 2.200 3.660 7.380 0.254 4 2.160 3.700 7.410 0.269 5 1.840 4.6307.960 0.292 5 1 2.150 4.300 9.140 2 1.980 3.680 8.480 3 2.220 4.2306.810 4 2.000 4.580 7.640 5 1.900 4.310 8.630 Summary Count 25 25 25 20Mean 2.050 4.114 7.960 0.226 SD 0.156 0.309 0.520 0.042 LOB 0.309 LOD0.392 LOQ 2.050

Example 10 Linearity and Assay Reference Interval

To establish the linearity of rT3 detection, five samples were preparedfrom different proportions of blank striped serum and striped serumspiked with 200 ng/dL. Two duplicates of each sample ranging from 0% to100% of the spiked serum were analyzed and the results plotted. A graphshowing the linearity of resulting curve is shown in FIG. 7.

Reference interval studies were conducted by analyzing samples from 115adults, including 61 females and 54 males between the ages of 18-86years. The inclusion criteria were: apparently healthy, ambulatory,community dwelling, non-medicated adults. The exclusion criteria werenormal TSH, FT4, FT3, anti-TPO and anti-TG, no history of chronicdisease, medication or recent medical problems. The resulting data wereanalyzed to develop a normal reference interval. Results are presentedin Table 5.

TABLE 5 Reference Interval rT3 (ng/dL) Reference Interval Lower Limit7.000 Reference Interval Upper Limit 26.000 Reference Interval Median15.000 Number of donors 115 Number above RI 5 Number below RI 3 Percentoutside RI 7%

Example 11 Sample Type Studies

Samples from thirty patients were collected in various Vacutainer® Tubesto result in serum, EDTA plasma, Heparin Plasma, and serum from SerumSeparation Tubes with gel barriers (i.e., SST sample tubes). Theresulting samples were analyzed and the results compared. All sampletypes were determined to be acceptable for clinical analysis. Comparisonplots of EDTA plasma, Heparin plasma, and SST serum samples versus serumare shown in FIGS. 8A, 9A, and 10A, respectively; difference plots areshown in FIGS. 8B, 9B, and 10B, respectively.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount ofreverse T3 (rT3) in a body fluid sample by mass spectrometry, saidmethod comprising: a. ionizing rT3 from the body fluid sample togenerate one or more reverse T3 ions detectable by mass spectrometry; b.determining the amount of one or more rT3 ions by mass spectrometry; andc. using the amount of said rT3 ions to determine the amount of rT3 inthe body fluid sample, wherein rT3 from the body fluid sample is notsubjected to solid phase extraction prior to ionizing.
 2. The method ofclaim 1, further comprising subjecting the rT3 from the body fluidsample to liquid chromatography prior to ionizing.
 3. The method ofclaim 2, wherein liquid chromatography comprises high performance liquidchromatography (HPLC).
 4. The method of claim 2, further comprisingsubjecting the further comprising enriching rT3 in said body fluidsample by protein precipitation prior to liquid chromatography.
 5. Themethod of claim 4, wherein said protein precipitation comprisescontacting said body fluid sample with methanol in an amount sufficientto precipitate at least a portion of proteins that may be present in thesample.
 6. The method of claim 1, further comprising enriching rT3 insaid body fluid sample by protein precipitation prior to ionizing. 7.The method of claim 6, wherein said protein precipitation comprisescontacting said body fluid sample with methanol in an amount sufficientto precipitate at least a portion of proteins that may be present in thesample.
 8. The method of claim 1, wherein the one or more rT3 ionsdetectable by mass spectrometry comprise one or more selected from thegroup consisting of ions with a mass/charge ratio of 649.9±0.5,605.2±0.5 and 127.1±0.5.
 9. The method of claim 1, wherein the one ormore rT3 ions detectable by mass spectrometry comprise one or moreselected from the group consisting of ions with a mass/charge ratio of649.9±0.5 and 605.2±0.5.
 10. The method of claim 1, wherein said massspectrometry is tandem mass spectrometry.
 11. The method of claim 10,wherein the one or more rT3 ions detectable by mass spectrometrycomprise a precursor ion with a mass/charge ratio of 649.9±0.5, and afragment ion selected from the group of ions with mass/charge ratio of605.2±0.5 and 127.1±0.5.
 12. The method of claim 11, wherein thefragment ion is an ion with mass/charge ratio of 605.2±0.5.
 13. Themethod of claim 1, wherein said body fluid sample comprises plasma orserum.
 14. A method for determining the amount of reverse T3 (rT3) in abody fluid sample by mass spectrometry, said method comprising: a.processing a body fluid sample to generate a processed sample comprisingrT3 from said body fluid sample; said processing comprising: i.precipitating protein from said body fluid sample by adding an organicsolvent, such that the resulting supernatant comprises said organicsolvent and rT3 from said body fluid sample; ii. purifying rT3 in saidsupernatant by subjecting said supernatant to a reverse-phase highperformance liquid chromatography (RP-HPLC) column; wherein saidpurifying comprises introducing an aqueous solution to said columnimmediately prior to introducing said supernatant; iii. eluting rT3 fromsaid RP-HPLC column to generate a processed sample comprising rT3; b.ionizing rT3 in said processed sample to generate one or more reverse T3ions detectable by mass spectrometry; c. determining the amount of oneor more rT3 ions by mass spectrometry; and d. using the amount of saidrT3 ions to determine the amount of rT3 in the body fluid sample. 15.The method of claim 14, wherein the ratio of the supernatant volumesubjected to said column to the aqueous plug volume introduce to thecolumn immediately prior is within the range of about 10:1 to 1:10. 16.The method of claim 14, wherein the ratio of the supernatant volumesubjected to said column to the aqueous plug volume introduce to thecolumn immediately prior is within the range of about 5:1 to 1:5. 17.The method of claim 14, wherein the ratio of the supernatant volumesubjected to said column to the aqueous plug volume introduce to thecolumn immediately prior is about 1:1.
 18. The method of claim 14,wherein said protein precipitation comprises contacting said body fluidsample with methanol in an amount sufficient to precipitate at least aportion of proteins that may be present in the sample.
 19. The method ofclaim 18, wherein said supernatant comprises at least 10% methanol. 20.The method of claim 14, wherein the one or more rT3 ions detectable bymass spectrometry comprise one or more selected from the groupconsisting of ions with a mass/charge ratio of 649.9±0.5, 605.2±0.5 and127.1±0.5.
 21. The method of claim 14, wherein the one or more rT3 ionsdetectable by mass spectrometry comprise one or more selected from thegroup consisting of ions with a mass/charge ratio of 649.9±0.5 and605.2±0.5.
 22. The method of claim 14, wherein said mass spectrometry istandem mass spectrometry.
 23. The method of claim 22, wherein the one ormore rT3 ions detectable by mass spectrometry comprise a precursor ionwith a mass/charge ratio of 649.9±0.5, and a fragment ion selected fromthe group of ions with mass/charge ratio of 605.2±0.5 and 127.1±0.5. 24.The method of claim 23, wherein the fragment ion is an ion withmass/charge ratio of 605.2±0.5.
 25. The method of claim 14, wherein saidbody fluid sample comprises plasma or serum.